The techniques of plant biotechnology have improved during the last ten years so that most of the crop species that are important to the mankind can be routinely transformed. The industry seeks for new traits not only for agricultural or nutritional purposes but as well for pharmaceutical purposes. There is an increasing interest to develop efficient and economic production systems for useful biological compounds. Transgenic plants are in the scope of research aiming to develop such a system. Given the concerns of environmental impacts of genetically modified crops this development has clearly created a need for a reliable system to prevent transgene flow among crops and in their relatives. Accordingly, several research groups around the world are currently engaged in developing techniques for gene containment in transgenic crops. The present invention discloses an improved transgene containment method and tools to achieve transgene containment. The method according to the present disclosure is superior in its reliability as compared to the systems that are known in the prior art.
Basically, the technologies that are aimed to prevent transgene flow can be categorized into one-component and two-component technologies. The main feature of the one-component systems is a possession of factor for negative selection of transgene from plant population. As an example there are well known concepts of male sterility, chloroplast transformation or ‘Terminator’ technology. The one-component systems are capable of decreasing gene flow but they do not provide an absolutely reliable containment. Therefore, one component systems might not give the wished result in situations where a total prevention of transgene flow is wanted.
The two-factor technologies are recently developed for improved transgene containment. Generally these systems use negative selection factors together with recovering (rescuing or repairing) factor. The negative selection factors are usually lethal for the plant and therefore they can absolutely prevent the transgene flow. The rescuing factor represses the action of the selection factor, disrupts its function or recovers the functions it blocked. Examples of two factor technologies are systems described in international patent publications WO 94/03619 (Bright et al.) and WO 00/37660 (Fabijanski et al).
International patent publication WO 02/064801 (Kuvshinov et al) describes a two-factor system, where excision construct (EC) is linked to the TGI. The EC excises the whole insert from the genome of the host organism under natural conditions. Artificially activated repression construct represses the action of the EC and saves the transgenic insert in the host genome. This system removes the entire transgene insert and leaves the host genome free from the foreign genes. Thus, in natural conditions transgenic plant produces non-transgenic seeds only and can not produce transgenic seeds.
Although the publications described above give advanced alternatives to control transgene flow, none of them resolves the problem of the negative selection gene being inactivated by mutagenesis or by silencing mechanism. According to Gressel (1999), such an inactivation of negative selection factor (gene) can happen approximately with a frequency of 10−6. In practice this means once in each middle sized field plot during a growth season. Such a frequency of gene escape for example from a field where the transgenic crop is cultivated for production of a vaccine or other pharmaceutical compound would create concerns of various parties.
This problem of inactivation has been proposed to be solved by one-component concept called mitigation tandem technique. In this technique the desired transgene is coupled in tandem with gene(s) that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Examples of features that could be used in mitigation technique are secondary dormancy and dwarfing. The problem encountered with the tandem mitigation technique is that due to absence of a recovering system removal of transgene from the population demands several generations and therefore this technique does not provide a system reliable enough for transgene containment. Another limitation of the technology is scarce sources of genes capable to mitigation.
U.S. patent application Ser. No. 09/617,543 (Kuvshinov et al.), now U.S. Pat. No. 6,849,776 discloses a two-factor system called RBF (recoverable block of function system) comprising at least one bClocking construct (BC) linked to a transgene of interest TGI and a recovering construct (RC). According to this disclosure BC blocks a vital physiological or molecular function of the host plant through developmental or organ specific expression. The RC is induced by an externally controllable stimulus and when induced it recovers the function previously blocked by expression of the BC(s). The present disclosure describes an improved RBF system.
The improved RBF system according to the present disclosure markedly decreases the probability of breach of the gene containment due to mutagenesis or silencing of the selection factor (i.e. the blocking sequence of the BC). The invention according to the present disclosure contains two blocking constructs. According to the present invention blocking nucleotides in the two blocking constructs may be different whereby the breach of the containment system becomes clearly less probable than in any of the previously described systems. The system includes a recovering mechanism whereby the progeny can carry the transgene of interest only under controlled recovery process.